POLYMERASE CHAIN REACTION: METHODS, PRINCIPLES ANDAPPLICATION

Dr.Mohini Joshi1*, Dr.Deshpande J.D2.

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. There are three major steps involved in the PCR technique: denaturation, annealing, and extension. PCR is useful in the investigation and diagnosis of a growing number of diseases. Qualitative PCR can be used to detect not only human genes but also genes of bacteria and viruses. PCR is also used in forensics laboratories and is especially useful because only a tiny amount of original DNA is required. PCR can identify genes that have been implicated in the development of cancer. Molecular cloning has benefited from the emergence of PCR as a technique. The present paper is an attempt to review basics of PCR.

Introduction

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Polymerase Chain Reaction was developed in 1984 by the American biochemist, Kary Mullis. Mullis received the Nobel Prize and the Japan Prize for developing PCR in 1993.1 However the basic principle of replicating a piece of DNA using two primers had already been described by Gobind Khorana in 1971. Progress was limited by primer synthesis and polymerase purification issues.2 PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.3 The polymerase chain reaction is a powerful technique that has rapidly become one of the most widely used techniques in molecular biology because it is quick, inexpensive and simple. The technique amplifies specific DNA fragments from minute quantities of source DNA material, even when that source DNA is of relatively poor quality.4 PCR; the quick, easy method for generating unlimited copies of any fragment of DNA, is one of those scientific developments that actually deserve timeworn superlatives like "revolutionary" and "breakthrough. From the daily practicalities of medical diagnosis to the

theoretical framework of systematics, from courts of law to field studies of animal behavior, PCR takes analysis of tiny amounts of genetic material-even damaged genetic material to a new level of precision and reliability. Furthermore, many important contributions to the development and application of PCR technology have been made; however the present paper is an attempt to review basics of PCR.

Basic Concept of PCR

The basic PCR principle is simple. As the name implies, it is a chain reaction: One DNA molecule is used to produce two copies, then four, then eight and so forth. This continuous doubling is accomplished by specific proteins known as polymerases, enzymes that are able to string together individual DNA Building blocks to form long molecular strands. To do their job polymerases require a supply of DNA building blocks, i.e. the nucleotides consisting of the four bases adenine (A), thymine (T), cytosine (C) and guanine (G). They also need a small fragment of DNA, known as the primer, to which they attach the building blocks as well as a longer DNA molecule to serve as a template for constructing the new strand. If these three ingredients are supplied, the enzymes will construct exact copies of the templates. PCR is a method used to acquire many copies of any particular strand of nucleic acids. It’s a means of selectively amplifying a particular segment of DNA. The segment may represent a small part of a large and complex mixture of DNAs e.g. a specific exon of a human gene. It can be thought of as a molecular photocopier. PCR can amplify a usable amount of DNA (visibel by gel electrophoresis) in ~2 hours. The template DNA need not be highly purified a boiled bacterial colony. The PCR product can be digested with restriction enzymes, sequenced or cloned. PCR can amplify a single DNA molecule, e.g. from a single sperm. The polymerase chain reaction relies on the ability of DNAcopying enzymes to remain stable at high temperatures. PCR has transformed the way that almost all studies requiring the manipulation of DNA fragments may be performed as a result of its simplicity and usefulness.5 In Mullis's original PCR process, the enzyme was used in vitro. The double-stranded DNA was separated into two single strands of DNA by heating it to 96°C. At this temperature, however, the E.Coli DNA polymerase was destroyed, so that the enzyme had to be replenished with new fresh enzyme rafter the heating stage of each cycle. Mullis's original PCR process was very inefficient since it required a great deal of time, vast amounts of DNA-Polymerase, and continual attention throughout the PCR process.

Steps in PCR

There are three major steps involved in the PCR technique: denaturation, annealing, and extension.

In step one; the DNA is denatured at high temperatures (from 90 - 97 degrees Celsius).

In step two, primers anneal to the DNA template strands to prime extension.

In step three, extension occurs at the end of the annealed primers to create a complementary copy strand of DNA.

This effectively doubles the DNA quantity through the third steps in the PCR cycle. To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called "Taq polymerase" synthesizes builds two new strands of DNA, using the original strands as templates. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and so on.7 The annealing phase happens at a lower temperature, 50-60°C. This allows the primers to hybridize to their respective complementary template strands, a very useful tool to forensik chemistry. The newly-formed DNA strand of primer attached to template is then used to create identical copies off the original template strands desired. Taq polymerase adds available nucleotides to the end of the annealed primers. The extension of the primers by Taq polymerase occurs at approx 72°C for 2-5 minutes. DNA polymerase I cannot be used to elongate the primers as one would expect because it is not stable at the high temperatures required for PCR. The beauty of the PCR cycle and process is that it is very fast compared to other techniques and each cycle doubles the number of copies of the desired DNA strand. After 25-30 cycles, whoever is performing the PCR process on a sample of DNA will have plenty of copies of the original DNA sample to conduct experimentation. Assuming the maximum amount of time for each step, 30 cycles would only take 6 hours to complete.

As the process of denaturation, annealing, and polymerase extension is continued the primers repeatedly bind to both the original DNA template and complementary sites in the newly synthesized strands and are extended to produce new copies of DNA. The end result is an exponential increase in the

total number of DNA fragments that include the sequences between the PCR primers, which are finally represented at a theoretical abundance of 2n, where n, is the number of cycles.

Due to the introduction of a thermostable DNA polymerase, the Taq DNA polymerase once, at the beginning of the PCR reaction.9 The thermostable properties of the DNA polymerase activity were isolated from Thermus aquaticus (Taq) that grow in geysers of over 110°C, and have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction. The Taq enzyme can withstand repeated heating to 94°C and so each time the mixture is cooled to allow the oligonucleotide primers to bind the catalyst for the extension is already present.10 After the last cycle, samples are usually incubated at 72°C for 5 minutes to fill in the protruding ends of newly synthesized PCR products. To ensure success, care should be taken both in preparing the reaction mixture and setting up the cycling conditions. Increasing the cycle number above ~35 has little positive effect because the plateau occurs when the reagents are depleted; accumulate. The specificity of amplification depends on the extent to which the primers can recognize and bind to sequences other than the intended target DNA sequences.

Basic Techniques in Molecular BiologyDr. Stefan Surzycki.

PCR Analysis

The Polymerase Chain Reaction (PCR) is a powerful method of in vitro DNA synthesis. Large amounts of a specific segment of DNA, of defined length and sequence, can be synthesized from a small amount oftemplate. PCRis a rapid, sensitive and inexpensive procedure for amplifying DNA of specific interest. The technique has revolutionized molecular biology and is used in virtually every area of natural sciences and medicine. The technique has cut across the boundaries separating basic and applied research, Commercial technology and medicine in a way few techniques ever have.

The principal ofPCRis rather simp le and involves enzymatic amplification of a DNA fragment flanked by two oligonucleotides (primers) hybridized to opposite strands of the template with the 3' ends facing each other (Figure 1). DNA polymerase synthesizes new DNA starting from the 3'end of each primer. Repeated cycles of heat denaturation of the template, annealing of the primers and extension of the annealed primers by DNApolymerase results in amplification of the DNA fragment. The extension products of each primer can serve as template for the other primer resulting in essentially doubling the amount of the DNA fragment in each cycle. The result is an exponential increase in the amount of specific DNA fragment defined by the 5' ends of the primers.

The PCRrevolution began quietly enough in the mind ofKary Mullis. By his own account, Mullis, then at Cetus Corporation, thought up the technique one Friday night in the summer of 1983(Mullis, 1990). The following winter, having made some educated guesses about concentrations and reaction times, he carried out his first experiment. He was lucky because the first time he tried the reaction, it worked! The initial procedure used Klenow fragment of E.coliDNApolymerase I added fresh during each cycle because the enzyme was inactivated by each denaturation step (Saiki et all 1985; Mullis et all 1986). The introduction of thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus (Taq) (Saiki et al. 1988) permitted the development of instruments that automated the Cycling portion of the procedure, substantially reducing the work needed to performa PCR.The use of this polymerase also increased the specificity and the yield of the desired

product because higher temperatures for annealing and extension could be used.

Since its introduction, numerous modifications and applications of the PCR technique have been developed, the presentation of even a small number of them vastly exceeds the scope of this book. There are books that are dedicated to this subject (Dieffenbach and Dveksler 1995; Innis et all 1995; Griffin and Griffin, 1994; Innis et al, 1990; White et all 1980). In this chapter, the most basic PCR concepts are presented along with general protocols. The aim is to provide an overall introduction to PCR technology that can serve as the basis for understanding more sophisticated applications.

Elements of standard PCR reactionComponents of a standard PCR reaction are: thermostable DNA polymerase, DNA template, primers, dNTP substrate, MgClz buffer and salt. In addition, PCR reactions frequently include compounds that stabilize the enzyme and reagents that help DNA dissociation or primer annealing.

DNA Polymerase

The most commonly used thermostable DNApolymerase is Taq polymerase. Consequently most standard PCRprotocols are optimized for maksimal activity of this enzyme. However, Taq polymerase exhibits some properties that make it less than ideal for some applications. First, the enzyme has very high error rates due to the lack of 3' to 5' exonuclease activity (Saiki et al. 1998). This may interfere with the preparation of DNA for sequencing, as well as with the inability to obtain long replication products. Second, the enzyme adds nucleotides to 3' ends in a template-independent manner, making the amplification product difficult to clone. Third, the enzyme is quite expensive.

For these reasons a variety of thermostable DNApolymerases from thermophilic or hyperthermophilic bacteria have been isolated, characterized and commercially introduced to PCR. Table 3 presents a list of these polymerases and their major characteristics. Some of these polymerases are used in standard PCR reactions (e.g., Tli, Pvo) while others are used in sequencing (e.g., Pvo, AmpliTherm) or in long PCR reaction mixtures (e.g., Tth).

The concentration of the enzyme in a reaction mixture can affect the fidelity of the product. An excessive amount of polymerase can result in amplification of nonspecific PCR products. The recommended amount of enzyme per reaction for most polymerases is 1 to 5 units. The most frequently used concentration is 2.0 units/lOO ~L1 reaction. However, the enzyme requirement may vary with respect to individual template DNA or primer used. It is always prudent to use the enzyme concentration recommended by the supplier since enzyme activity from different manufacturer can vary.

DNA Template

One of the most important features of PCR is that it can be performed with very small quantities of relatively impure DNA.Even degraded DNAcan be successfully amplified. Therefore a number of simple and rapid protocols to purify DNAfor PCRhave been developed. In this book some of these methods are described in chapters 2, 3,4 and 5about DNApurification. In spite of the fact that DNA need not be absolutely pure, a number of contaminants can decrease the efficiency of amplification. The presence of urea, SDS, sodium acetate and some components eluted from agarose can interfere with PCR.Most of these impurities can be removed by washing with 70%ethanol or by reprecipitation of DNA in the presence of ammonium acetate. Protocols for removal of these contaminants are described in this book in the chapters on DNA purification.

Primers

General guidelines for selection of primers for PCR are:

• Optimal primer set should hybridize to the template efficiently with negligible hybridization to other sequences of the sample. To assure this, primers should be at least 20 to 25 bases in length. However, for some applications requiring multiple random initiation (e.g., RAPD analysis), primers are usually 8 to 10 bases in length.

• Primers, if possible, should have a GCcontent similar to that of the target. Overall GCcontent of 40%to 60% usually works for most reactions. The GC content of both primer should be similar.

• Primers should not have sequences with significant secondary structure. Therefore, primer sequence should not contain simple repeats or palindromic sequences.• Primer pairs should not contain complementary sequences to Beach other. In particular primers with 3' overlaps should be avoided. This will reduce the incidence of "primer-dimer" formation that severely affects efficiency of amplification.

Many commercial computer programs exist to design primer sets. Some excellent freely shared programs are: PRIMER from Whitehead Institute for Biomedical Research (http://www-genome.wi.edu) for PC Computer and Amplify and HyperPCR for Macintosh computers (ftp://iubio.bio.indiana. edu.)

The PCR reaction requires a molar excess of primers. Primer concentration is usually 0.2 to 1 J..lM. Using too much primer may resu lt in false initiations and "primer-dimer" formation.

The distance between primers can vary considerably. However, the efficiency of amplification decreases considerably for distances greater than 3 kbp. In general, short fragments amplify with higher efficiency than long ones. Preferential amplification of short fragments is probably due to more efficient full-length extension by the enzyme in each cycle. Amplification of a very large fragment is possible with modifications of standard reaction conditions and by using a mixture of DNA polymerases (e.g., Taq and Pfu). These conditions are referred to as long PCR. Numerous commercial kits exist that use proprietary reagents to achieve long PCR.

Substrate

The concentration of each of dNTP in PCRshould not exceed 200IlM. This amount of substrate is sufficient to synthesize 12.5ug of DNAwhen half of the nucleotides are incorporated, a quantity far exceeding the amount of DNA synthesized in standard PCR (see Table 1). The four dNTPs should be used at equivalent concentrations, particularly when Taq polymerase is used. This will minimize the error rate of the enzyme. An excess of nucleotides inhibits enzyme activity and can contribute to the appearance of false products. Moreover, when varying dNTP concentration, it should be remembered that dNTPs chelate magnesium ion, decreasing its concentration in the reaction mixture.

MgCl2 Concentration

Magnesium ion is a required co-factor for all DNApolymerases. Moreover magnesium ion concentration may affect the following:

• Primer annealing.• Temperature of strand dissociation for template and product.• Product specificity.• Formation of primer-dimer artifacts and enzyme fidelity.

Many templates require optimization of magnesium ion concentration for efficient, correct amplification. The optimal concentrations of Mg++ for each thermophilic polymerase are listed in Table 3. They should serve as a starting point for optimizing the magnesium ion concentration in the reaction.

Buffer and Salt

A standard buffer for PCR is 10 to 50 mM Tris-HCl. The optimum pH is between 8 and 9 for most thermophilic polymerases (Table 3). Since the L1 pKa for Tris is high (- 0.031 jOC), the true pH of the reaction mixture during a typical thermal cycle varies considerably (approximately 1 to 1.5 pH units).

The salt used in most reactions is potassium or sodium. The K+ (Na") is usually added to facilitate correct primer annealing. For Taq polymerase, the concentration usually used is 50mM since higher monovalent ion concentrations inhibit polymerase activity (Innis et al. 1988).It is not necessary to use salt when amplifying DNA using a rapid cycle amplification instrument, since fidelity of primer annealing is achieved as a result of the short annealing time. Some other components used in reactions to help stabilize the enzyme are: gelatin, bovine serum albumin or nonionic detergent such as Tween 20or Triton X100. However, most protocols work well without the addition of these ingredients.

Thermal cycling profil

Standard PCR consists of three steps : denaturation, annealing and extension. These steps are repeated or cycled 25 to 30 times. Most protocols also include a single denaturation step (init ial denaturation) before cycling begins and single long extension step (final extension) at the end.

An initial denaturation that lasts for a few minutes is added to assure complete denaturation of the template. This preliminary step is important for complex templates or templates with high GCcontent. The final extension step is usually carried out for 5 to 10 minutes to assure completion of partial extension products by DNA polymerase and to provide time for complete renaturation of single stranded products.

Denaturation Step

In this step, complete separation of the template strands must be accomplished. Denaturation is a first order reaction that occurs very fast. A complete description of the denaturation reaction is given in chapter 10.Astan dard time 000 to 60 seconds is used to assure uniform heating of the entire reaction volume. When a rapid cycle instrument is used, this time is usually set to 0 for a 10ul reaction cycled in a glass capillary tube. The temperatur of cycle denaturation is usually 94 °C to 95 "C.

Annealing Step

In this step, primers anneal to the template. With excess primer the annealing reaction is pseudo first order reaction (see chapter 10 for an explanation) and occurs very fast. The time used for this step, in most protocols, is 30 to 60 seconds, the time required to cool the reaction from the denaturation temperature to the annealing temperature. In a rapid cycle instrument, this time is usually set to O. The annealing temperature depends on the GC content of the primers and should be carefully adjusted . Lowannealing temperatures will result in unspecific primer annealing and initiation. High temperatures will prevent annealing and consequently will prevent DNA synthesis. For most primers, an annealing temperature of 50 °C to 55 °C is used without furthe r optimization.

Elongation Step

In this step, DNA polymerase synthesizes a new DNA strand by extending the 3' ends of the primers. Time of the elongation depends on the length of the sequence to be amplified . Since Taq polymerase can add 60-100 bases per second under optimal conditions, synthesis of a 1 Kbp fragment should require a little less than 20 seconds. However, most protocols recommend 60 seconds per 1 Kbp DNAto account for time needed to reach the correct temperature and to compensate for other unknown factors that can affect reaction rate . The shortest possible time should be used to preserve polymerase activity. In air rapid cycle instruments, the elongation time is usually set to that calculated for the theoretical elongation rate of polymerase (15 to 20 seconds per 1 Kbp of desired fragment) .

An extension temperature of 72 °C is used in standard amplification protocols. This temperature is close to the optimal temperature for Taq polymerase (75 °C) but low enough to prevent dissocia tion of the primer from the template. However, it should be remembered that Taq polymerase has substantial elongation activity at 55 °C, the temperature used in most annealing steps . This residual synthesis stabilizes the interaction of primer with template and indeed permits the use of an elongation temperatur higher than 72 "C, Higher temperatures of elongation are used when the template can form secondary structures that interfere with elongation.

Principles and Technique of Biochemistry and Molecular BiologyWilson and Walker

Basic Concept of the PCR

The polymerase chain reaction or PCR is one of the mainstays of molecular biology. One of the reasons for the wide adoption of the PCR is the elegant simplicity of the reaction and relative ease of the practical manipulation steps. Indeed combined with the relevant bioinformatics resources for its design and for determination of the required experimental conditions it provides a rapid means for DNA identification and analysis. It has opened up the investigation of cellular and molecular processes to those outside the field of molecular biology.

The PCR is used to amplify a precise fragment of DNA from a complex mixture of starting material usually termed the template DNA and in many cases requires little DNA purification. It does require the knowledge of some DNA sequence information which flanks the fragment of DNA to be amplified (target DNA). From this information two oligonucleotide primers may be chemically synthesised each complementary to a stretch of DNA to the 30 side of the target DNA, one oligonucleotide for each of the two DNA strands (Fig. 5.32). It may be thought of as a technique analogous to the DNA replication process that takes place in cells since the outcome is the same: the generation of new complementary DNA stretches based upon the existing ones. It is also a technique that has replaced, in many cases, the tradisional DNA cloning methods since it fulfils the same function, the production of large amounts of DNA from limited starting material; however, this is achieved in a fraction of the time needed to clone a DNA fragment (Chapter 6). Although not without its drawbacks the PCR is a remarkable development which is changing the approach of many scientists to the analysis of nucleic acids and continues to have a profound impact on core biosciences and biotechnology.

Stages in PCR

The PCR consists of three defined sets of times and temperatures termed steps: (i) denaturation, (ii) annealing and (iii) extension. Each of these steps is repeated 30–40 times, termed cycles (Fig. 5.33). In the first cycle the double-stranded template DNA is (i) denatured by heating the reaction to above 90 _C. Within the complex DNA the region to be specifically amplified (target) is made accessible. The temperature is then cooled to 40–60 _C. The precise temperature is critical and each PCR system has to be defined and optimised. One useful technique for optimisation is touchdown PCR where a programmable cycler is used to incrementally decrease the annealing temperature until the optimum is derived. Reactions that are not optimised may Gide rise to other DNA products in addition to the specific target or may not produce any amplified products at all. The annealing step allows the hybridisation of the two oligonucleotide primers, which are present in excess, to bind to their complementary sites that flank the target DNA. The annealed oligonucleotides act as primers for DNA synthesis, since they provide a free 30 hydroxyl group for DNA polymerase. The DNA

synthesis step is termed extension and is carried out by a thermostable DNA polymerase, most commonly Taq DNA polymerase.

DNA synthesis proceeds from both of the primers until the new strands have been extended along and beyond the target DNA to be amplified. It is important to note that, since the new strands extend beyond the target DNA, they will contain a region near their 30 ends that is complementary to the other primer. Thus, if another round of DNA synthesis is allowed to take place, not only the original strands will be used as templates but also the new strands. Most interestingly, the products obtained from the new strands will have a precise length, delimited exactly by the two regions complementary to the primers. As the system is taken through successive cycles of denaturation, annealing and extension all the new strands will act as templates and so there will be an exponential increase in the amount of DNA produced. The net effect is to selectively amplify the target DNA and the primer regions flanking it (Fig. 5.34).

One problem with early PCR reactions was that the temperature needed to denature the DNA also denatured the DNA polymerase. However the availability of a thermostable DNA polymerase enzyme isolated from the thermophilic bacterium Thermus aquaticus found in hot springs provided the means to automate the reaction. Taq DNA polymerase has a temperature optimum of 72 _C and survives prolonged exposure to temperatures as high as 96 _C and so is still active after each of the denaturation steps. The widespread utility of the technique is also due to the ability to automate the reaction and as such many thermal cyclers have been produced in which it is possible to program in the temperatures and times for a particular PCR reaction.

Molecular Biotechnology

Principles and Applications of Recombinant DNA

Glick, Pasternak, and Patten

Polymerase Chain Reaction

PCR is an effective procedure for generating large quantities of a specific DNA sequence in vitro. This amplification, which can be more than a millionfold, is achieved by a three-step cycling process. The essential compo nents for PCR amplification are (1) two synthetic oligonucleotide primer (~20 nucleotides each) that are complementary to region on opposite strands that flank the target DNA sequence and that, after annealing to the source DNA, have their 3′ hydroxyl ends oriented toward each other; (2) a template sequence in a DNA sample that lies between the primer-binding sites and that can be from 100 to ~35,000 bp in length; (3) a thermostable DNA polymerase that can withstand being heated to 95°C or higher and that copies the DNA template with high fidelity; and (4) the four deoxyribonucleotides.

A typical PCR process entails a number of cycles for amplifying a specific DNA sequence. Each cycle has three successive steps.

1. Denaturation. The first step in the PCR amplification system is the thermal denaturation of the DNA sample by raising the temperatur within a reaction tube to 95°C. In addition to the source template DNA, this reaction tube contains a vast molar excess of the two oligonucleotide primers, a thermostable DNA polymerase (e.g., Taq DNA polymerase, isolated from the bacterium Thermus aquaticus), and four deoxyribonucleotides. The temperature is maintained for about 1 minute.

2. Renaturation. For the second step, the temperature of the mixture is slowly lowered to ~55°C. During this step, the primers base pair with their complementary sequences in the DNA sample.

3. Synthesis. In the third step, the temperature is raised to ~75°C, which is optimum for the catalytic functioning of Taq DNA polymerase. DNA synthesis is

initiated at the 3 hydroxyl end of each primer and uses the source DNA as a ′template (Fig. 4.14).

All steps in a PCR cycle are carried out in an automated block heater that is programmed to change temperatures after a specified period of time. One cycle generally lasts from 3 to 5 minutes.

To understand how the PCR protocol succeeds in amplifying a discrete segment of DNA, it is important to keep in mind the location of Beach primer-annealing site and its complementary sequence within the strands that are synthesized during each cycle. During the synthesis phase of the first cycle, the newly synthesized DNA from each primer is extended beyond the endpoint of the sequence that is complementary to the second primer. These new strands form “long templates” that are used in the second cycle (Fig. 4.14).

During the second cycle, the original DNA strands and the new strands synthesized in the first cycle (long templates) are denatured and then hybridized with the primers. The large molar excess of primers in the reaction mixture ensures that they will hybridize to the template DNA before complementary template strands have the chance to reanneal to each other. A second round of synthesis produces long templates from the original strands, as well as some DNA strands that have a primer sequence at one end and terminate with a sequence complementary to the other primer at the other end (“short templates”) that were generated from the long templates (Fig. 4.15).

During the third cycle, short templates, long templates, and original strands all hybridize with the primers and are replicated (Fig. 4.16). In subsequent cycles, the short templates preferentially accumulate, and by the 30th cycle, these strands are about a million times more abundant than either the original or long template strands (Fig. 4.17). PCR has become a pervasive technique that is used for innumerable purposes, some of which are described here and many others in the ensuing chapters.

Gene Cloning and DNA AnalysisChapter 9 The Polymerase Chain Reaction

The PCR outlineThe polymerase chain reaction results in the selective amplification of a chosen region of a DNA molecule. Any region of any DNA molecule can be chosen, so long as the sequences at the borders of the region are known. The border sequences must be known because in order to carry out a PCR, two short oligonucleotides must hybridize to the DNA molecule, one to each strand of the double helix (Figure 9.1). These oligonucleotides, which act as primers for the DNA synthesis reactions, delimit the region that will be amplified.

Amplification is usually carried out by the DNA polymerase I enzyme from Thermus aquaticus. As mentioned on p. 49, this organism lives in hot springs, and many of its enzymes, including Taq polymerase, are thermostable, meaning that they are resistan to denaturation by heat treatment. As will be apparent in a moment, the thermostability of Taq polymerase is an essential requirement in PCR methodology.

To carry out a PCR experiment, the target DNA is mixed with Taq polymerase, the two oligonucleotide primers, and a supply of nucleotides. The amount of target DNA can be very small because PCR is extremely sensitive and will work with just a singlet starting molecule. The reaction is started by heating the mixture to 94°C. At this temperature the hydrogen bonds that hold together the two polynucleotides of the double helix are broken, so the target DNA becomes denatured into single-stranded molecules (Figure 9.2). The temperature is then reduced to 50–60°C, which results in some rejoining of the single strands of the target DNA, but also allows the primers to attach to their annealing positions. DNA synthesis can now begin, so the temperature is raised to 74°C, just below the optimum for Taq polymerase. In this first stage of the PCR, a set of “long products” is synthesized from each strand of the target DNA. These polynucleotides have identical 5 ′ ends but random 3 ′ ends, the latter representing positions where DNA synthesis terminates by chance.

The cycle of denaturation–annealing–synthesis is now repeated (Figure 9.3). The long products denature and the four resulting strands are copied during the DNA synthesis stage. This gives four double-stranded molecules, two of which are identical to the long products from the first cycle and two of which are made entirely of new DNA. During the third cycle, the latter give rise to “short products”, the 5 ′ and 3 ′ ends of which are both set by the primer annealing positions. In subsequent cycles, the number of short products accumulates in an exponential fashion (doubling during each cycle) until one of the components of the reaction becomes depleted. This means that after 30 cycles, there will be over 130 million short products derived from each starting molecule. In real terms, this equates to several micrograms of PCR product from a few nanograms or less of target DNA.

At the end of a PCR a sample of the reaction mixture is usually analyzed by agarose gel electrophoresis, sufficient DNA having been produced for the amplified fragment to be visible as a discrete band after staining with ethidium bromide. This may by itself provide

useful information about the DNA region that has been amplified, or alternatively the PCR product can be examined by techniques such as DNA sequencing.

Designing the oligonucleotide primers for a PCR

The primers are the key to the success or failure of a PCR experiment. If the primer are designed correctly the experiment results in amplification of a single DNA fragment, corresponding to the target region of the template molecule. If the primers are incorrectly designed the experiment will fail, possibly because no amplification occurs, or possibly because the wrong fragment, or more than one fragment, is amplified (Figure 9.4). Clearly a great deal of thought must be put into the design of the primers.

Working out appropriate sequences for the primers is not a problem: they must correspond with the sequences flanking the target region on the template molecule. Each primer must, of course, be complementary (not identical) to its template strand in order for hybridization to occur, and the 3 ′ ends of the hybridized primers should point toward one another (Figure 9.5). The DNA fragment to be amplified should not be greater than about 3 kb in length and ideally less than 1 kb. Fragments up to 10 kb can be amplified by standard PCR techniques, but the longer the fragment the less efficient the amplification and the more difficult it is to obtain consistent results. Amplification of very long fragments—up to 40 kb—is possible, but requires special methods.

The first important issue to address is the length of the primers. If the primers are too short they might hybridize to non-target sites and give undesired amplification products. To illustrate this point, imagine that total human DNA is used in a PCR experiment with a pair of primers eight nucleotides in length (in PCR jargon, these are called “8-mers”). The likely result is that a number of different fragments will be amplified. This is because attachment sites for these primers are expected to occur, on average, once every 48 = 65,536 bp, giving approximately 49,000 possible sites in the 3,200,000 kb of nucleotide sequence that makes up the human genome. This means that it would be very unlikely that a pair of 8-mer primers would give a single, specific amplification product with human DNA (Figure 9.6a).

What if the 17-mer primers shown in Figure 9.5 are used? The expected frequency of a 17-mer sequence is once every 417 = 17,179,869,184 bp. This figure is over five times greater than the length of the human genome, so a 17-mer primer would be expected to have just one hybridization site in total human DNA. A pair of 17-mer primers should therefore give a single, specific amplification product (Figure 9.6b).

Why not simply make the primers as long as possible? The length of the primer influences the rate at which it hybridizes to the template DNA, long primers hybridizing at a slower rate. The efficiency of the PCR, measured by the number of amplified molecules produced during the experiment, is therefore reduced if the primers are too long, as complete hybridization to the template molecules cannot occur in the time allowed during the reaction cycle. In practice, primers longer than 30-mer are rarely used.

Working out the correct temperatures to use

During each cycle of a PCR, the reaction mixture is transferred between three temperatures (Figure 9.7):

l The denaturation temperature, usually 94°C, which breaks the base pairs and releases single-stranded DNA to act as templates in the next round of DNA synthesis;

l The hybridization or annealing temperature, at which the primers attach to the templates;

l The extension temperature, at which DNA synthesis occurs. This is usually set at 74°C, just below the optimum for Taq polymerase.

The annealing temperature is the important one because, again, this can affect the specificity of the reaction. DNA–DNA hybridization is a temperature-dependent phenomenon. If the temperature is too high no hybridization takes place; instead the primers and templates remain dissociated (Figure 9.8a). However, if the temperature is too low, mismatched hybrids—ones in which not all the correct base pairs have formed—are stable (Figure 9.8b). If this occurs the earlier calculations regarding the appropriate lengths for the primers become irrelevant, as these calculations assumed that only perfect primer–template hybrids are able to form. If mismatches are tolerated, the number of potential hybridization sites for each primer is greatly increased, and amplification is more likely to occur at non-target sites in the template molecule.

The ideal annealing temperature must be low enough to enable hybridization between primer and template, but high enough to prevent mismatched hybrids from forming (Figure 9.8c). This temperature can be estimated by determining the melting temperature or Tm of the primer–template hybrid. The Tm is the temperature at which the correctly base-paired hybrid dissociates (“melts”). A temperature 1–2°C below this should be low enough to allow the correct primer–template hybrid to form, but too high for a hybrid with a single mismatch to be stable. The Tm can be determined experimentally but is more usually calculated from the simple formula (Figure 9.9):

Tm = (4 × [G + C]) + (2 × [A + T])°C

in which [G + C] is the number of G and C nucleotides in the primer sequence, and [A + T] is the number of A and T nucleotides.

The annealing temperature for a PCR experiment is therefore determined by calculating the Tm for each primer and using a temperature of 1–2°C below this figure. Note that this means the two primers should be designed so that they have identical Tms. If this is not the case, the appropriate annealing temperature for one primer may be too high or too low for the other member of the pair.